Protein purification

Abstract

The present invention relates to a process for purification a protein comprising a semi-continuous chromatography step whereby the flow-through is collected and re-loaded onto the chromatography matrix.

I claim: 
     
       1. A process for the purification of a protein of interest from a mixture comprising the steps of
 a) in an operational chromatography cycle loading a first volume of a mixture containing the protein of interest from a first container to a chromatography matrix operated such that the protein of interest binds to the chromatography matrix until 40% to 100% of the maximum static binding capacity of the chromatography matrix for the protein of interest is reached; 
 b) collecting flow-through containing unbound protein of interest in a second container, and 
 c) in a further operational chromatography cycle re-loading the flow-through collected in the second container to the same chromatography matrix followed by loading a second volume of the protein of interest from the first container to the same chromatography matrix, operated such that the protein of interest binds to the chromatography matrix until 40% to 100% of the maximum static binding capacity of the chromatography matrix for the protein of interest is reached. 
 
     
     
       2. The process according to  claim 1 , wherein the further operational chromatography cycle immediately follows the first operational chromatography cycle. 
     
     
       3. The process according to  claim 1 , wherein the collection of the flow-through is started at a predetermined first concentration of protein of interest in the flow-through and stopped at a predetermined second concentration of protein of interest in the flow-through. 
     
     
       4. The process according to  claim 1 , wherein a predetermined fraction of the flow-through is collected in the second container. 
     
     
       5. The process according to  claim 1 , wherein the flow-through is processed prior to being re-loaded onto the chromatography matrix, the processing being selected from stirring or agitation, dilution, concentration adjustment, pH adjustment, conductivity adjustment, buffer or solvent exchange, cooling or heating and any combination thereof. 
     
     
       6. The process according to  claim 1 , wherein the operational chromatography cycle is selected from affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, chiral chromatography and dielectric chromatography. 
     
     
       7. The process according to  claim 1 , wherein the process comprises three chromatography steps. 
     
     
       8. The process according to  claim 7 , wherein the process comprises Protein A chromatography followed by cation exchange chromatography followed by anion exchange chromatography. 
     
     
       9. The process according to  claim 1 , wherein the chromatography matrix is a chromatography column comprising the chromatography matrix. 
     
     
       10. The process according to  claim 1 , wherein the protein of interest is an antibody or an antibody fragment. 
     
     
       11. A process for the purification of a protein of interest from a mixture comprising the steps of:
 a) in an operational chromatography cycle loading a first volume of a mixture containing the protein of interest from a first container to a chromatography matrix operated such that the dynamic binding capacity of the chromatography matrix for the protein of interest is exceeded; 
 b) collecting flow-through containing unbound protein of interest in a second container, and 
 c) in a further operational chromatography cycle re-loading the flow-through collected in the second container to the same chromatography matrix followed by loading a second volume of the protein of interest from the first container to the same chromatography matrix, operated such that the dynamic binding capacity of the chromatography matrix for the protein of interest is exceeded. 
 
     
     
       12. The process according to  claim 11 , wherein in step (a) the loading of the protein of interest is stopped when at least 40% of the maximum static binding capacity is reached. 
     
     
       13. The process according to  claim 11 , wherein the further operational chromatography cycle immediately follows the first operational chromatography cycle. 
     
     
       14. The process according to  claim 11 , wherein the collection of the flow-through is started at a predetermined first concentration of protein of interest in the flow-through and stopped at a predetermined second concentration of protein of interest in the flow-through. 
     
     
       15. The process according to  claim 11 , wherein a predetermined fraction of the flow-through is collected in the second container. 
     
     
       16. The process according to  claim 11 , wherein the flow-through is processed prior to being re-loaded onto the chromatography matrix, the processing being selected from stirring or agitation, dilution, concentration adjustment, pH adjustment, conductivity adjustment, buffer or solvent exchange, cooling or heating or any combination thereof.